The invention relates to an enzyme detection method and to its use in detection of parasites.
Protozoan parasites have an adverse effect on the health of human and animal populations in a large number of countries. Problems presented by protozoan parasites are particularly of concern in tropical areas of the world where modern diagnostic methods are often not easily accessible.
Nucleoside hydrolases are enzymes which hydrolyse nucleosides between the ribose or deoxyribose and the purine or pyrimidine base groups. A number of different types of nucleoside hydrolases are known. N-Ribohydrolases hydrolyse ribonucleosides and N-deoxyribohydrolases hydrolyse deoxyribonucleosides. Within each of these two general groups of enzymes are enzymes of differing specificities.
Among the well-characterized N-ribohydrolases, specificity is high for the ribosyl group but varies for the leaving group purine or pyrimidine. The inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from the trypanosome Crithidia fasciculata hydrolyzes all of the naturally occurring purine and pyrimidine nucleosides with similar catalytic efficiencies. The guanosine-inosine enzyme (GI-nucleoside hydrolase) from the same organism has a strong preference for the eponymous substrates and is nearly inert with the pyrimidine nucleosides1,2,3,4. AMP Nucleosidase from bacterial sources is highly specific for the adenine base, and the 5xe2x80x2-phosphoryl is required for significant hydrolytic rates5,6. Nucleoside phosphorylases have a similar mechanism to nucleoside hydrolases and, for example, purine nucleoside phosphorylase is specific for inosine and guanosine substrates and activates phosphate or arsenate anions to attack C1 of the nucleosides.
It is an object of the invention to provide a method of detecting and/or assaying for the presence of certain enzymes, especially those of parasites in samples taken from parasitised humans and animals.
In one aspect the invention provides a method of detecting and/or assaying nucleoside hydrolases using a chromogenic substrate.
Preferably the chromogenic substrates have the formula: 
where X is OH or H, and Y is the residue of Yxe2x80x94OH where Yxe2x80x94OH is a chromophore or a compound readily converted to a chromophore and the substrates are hydrolysed by the nucleoside hydrolase to yield ribose or 2-deoxyribose plus Yxe2x80x94OH.
Preferably the chromogenic substrates are of Formula I wherein X and Y are as defined and the substrates are phosphorylysed by the nucleoside phosphorylase to yield ribose-1-phosphate or 2-deoxyribose-1-phosphate plus Yxe2x80x94OH.
Y may be chosen so that Yxe2x80x94OH is a compound absorbing in the visible or TV light, readily measured at wavelengths greater than 300 nm, preferably greater than 340nm.
Preferably Yxe2x80x94OH is 6-hydroxynicotinamide or 2-hydroxypyridine -4-carboxamide.
Y may be chosen so that Yxe2x80x94OH is a chemiluminescent compound eg luminol which when released and oxidized by chemical or enzymatic means emits light.
Alternatively Yxe2x80x94OH may be a compound readily converted to a coloured compound eg by reaction with a diazonium salt eg xcex1-naphthol.
More preferably Yxe2x80x94OH is a fluorescent compound eg 4-methylumbelliferone or fluoroscein.
Most preferably Yxe2x80x94OH is a coloured compound eg phenolphthalein, p-nitrophenol, thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine.
It is preferred that Yxe2x80x94OH can be measured in the presence of the compound of Formula I by virtue of Yxe2x80x94OH absorbing or fluorescing to a greater extent than the compound of Formula I at certain wavelengths. Generally this will be by virtue of Yxe2x80x94OH ionising and being in equilibrium with quinonoid-like forms.
A particularly preferred substrate is p-nitrophenyl xcex2-D-ribofuranoside. Also particularly preferred is 4-pyridyl xcex2-D-ribofuranoside. Further particularly preferred substrates are 4-methylumbelliferyl xcex2-D-ribofuranoside (4-methylcoumarin-7-yl xcex2-D-ribofuranoside) and 2-(5-nitropyridyl) xcex2-D-ribofuranoside.
According to another aspect of the invention, there is provided a method for detecting and/or assaying for parasites especially protozoa in samples obtained usually from humans or animals using a chromogenic substrate, preferably of Formula I.
More preferably the chromogenic substrate is one where Yxe2x80x94OH is fluorescent or coloured.
Another preferred type of substrate is one where Yxe2x80x94OH is chemiluminescent eg luminol.
According to a further aspect of the invention there is provided a kit containing materials for detection or assay of hydrolysis of the chromogenic substrate, preferably of Formula I, by enzymes in a biological sample. Preferred kits comprise the chromogenic substrate in dry form, together with a buffer. Other components eg a cell-lysing agent may also be included.
According to a further aspect of the invention there is provided a dipstick containing a chromogenic substrate, preferably of Formula I, for use in detecting nucleoside hydrolases.
According to a further aspect of the invention there are provided novel compounds of the invention of formula I. In this aspect of the invention X and Y are as previously defined except the known compounds xcex1-naphthyl xcex2-D-ribofuranoside, 4-methylcoumarin-7-yl 2-deoxy-p-xcex2-ribofuranoside, xcex2-nitrophenyl xcex2-D-ribofuranoside, p-aminophenyl xcex2-D-ribofuranoside, 5-amino-6-chloro-3-pyridazinyl xcex2-D-ribofuranoside, 4-chlorophenyl xcex2-D-ribofuranoside, phenyl xcex2-D-ribofuranoside, 4-methoxyphenyl xcex2-D-ribofuranoside, 4-hydroxyphenyl xcex2-D-ribofuranoside, 4-(N,N,N-trimethylammonio )phenyl xcex2-D-ribofuranoside, 4-acetylphenyl xcex2-D-ribofuranoside and the xcex2-D-ribofuranosides of L-DOPA and L-xcex1-methyl-DOPA and the N-acetyl methyl esters of the abovementioned DOPA derivatives are not included in this aspect. The exceptions are not known as chromogenic substrates or for use in assay of parasites. Generally the chromogenic group is not coloured when present in the compound of Formula I but is readily detectable when that compound is hydrolysed to give Yxe2x80x94OH.
Preferred compounds are those defined above in which Y is an optionally substituted pyridyl group or a nitrophenyl group.
Particularly preferred novel compounds of the invention include 3-trifluoroacetamidophenyl xcex2-D-ribofuranoside, 3-aminophenyl- xcex2-D-ribofuranoside, 1-tetralone-5-yl xcex2-D-ribofuranoside, 3-( 4-hydroxyphenyl)-1-(3H)-isobenzofuranone-3-(phen-4-yl) xcex2-D-ribofuranoside, 2-nitrophenyl xcex2-D-ribofuranoside, 4-methylcoumarin-7-yl xcex2-D-ribofuranoside, 3-pyridyl xcex2-D-ribofuranoside, 4-pyridyl xcex2-D-ribofuranoside, 2-(5-nitropyridyl) xcex2-D-ribofuranoside, 5-quinolyl xcex2-D-ribofuranoside, the xcex2-D-ribofuranoside of luminol, p-nitrophenyl 2-deoxy- xcex2-D-erythro-pentofuranoside, 3-carboxamido-6-pyridyl xcex2-D-ribofuranoside, and 4-forymylphenyl xcex2-D-ribofuranoside.
In a further aspect of the invention there is provided a method of preparing a chromogenic substrate of the invention.
According to a further aspect the invention may be directed to a method to detect or assay for the presence of nucleoside phosphorylases using a chromogenic substrate.
One group of preferred chromogenic substrates have a xcex2-D-ribofuranosyl group attached through a xcex2-O-ribosidic linkage to a chromogenic group.
In a preferred embodiment the chromogenic substrate is a compound of Formula I wherein Xxe2x95x90OH. The group Y is as defined under Formula I. Preferably the chromophore Yxe2x80x94OH is selected from the group of phenolphthalein, p-nitrophenol, 4-methylumbelliferone, xcex1-naphthol, thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine, 6-hydroxynicotinamide, 2-hydroxypyridine-4-carboxamide and fluorescein. Other chromophores as will be known in the art may also be used.
In this specification the term xe2x80x9cchromogenic groupxe2x80x9d is used to refer to a group in a nucleoside hydrolase substrate bound to the sugar moiety which, when enzymnatically removed from the substrate, forms a compound which is readily detectable by its visible or UV absorption or by its fluorescence. It may be detectable either as released or after pH alteration, or after a subsequent reaction. The term xe2x80x9cchromophorexe2x80x9d is used for the readily detectable product as released and/or as detectable. The term xe2x80x9cchromogenxe2x80x9d as used in chemical names, eg 1-O-chromogen derivatives, indicates that the relevant group is a xe2x80x9cchromogenic groupxe2x80x9d as defined above. It is preferred that the xe2x80x9cchromophorexe2x80x9d is readily detectable by its visible absorption, or alternatively, by its fluorescence, but the definition herein also includes other molecules. The term xe2x80x9cchromogenic substratexe2x80x9d is used to refer to a substrate containing a xe2x80x9cchromogenic groupxe2x80x9d.
In general it is preferred that the chromophore Yxe2x80x94OH is coloured or can be converted to a coloured form simply by altering the pH. Especially preferred chromophores include p-nitrophenol, phenolphthalein, thymolphthalein and 2-hydroxy-5-nitropyridine. For applications where the enzyme levels are low, a fluorescent chromophore may be preferred, especially 4-methylumbelliferone.
The invention is not limited solely to the detection of nucleoside hydrolases. Nucleoside phosphorylases are not hydrolases as they use phosphate as the nucleophile rather than water. The result is a yield of ribose-l-phosphate rather than ribose. Nucleoside phosphorylases have a similar mechanism to nucleoside hydrolases and can be detected using the method of the present invention.
On hydrolysis the chromogenic group is released from the molecule and may be conveniently measured as the chromophore. Phenolphthalein released by hydrolysis gives a red colour, p-nitrophenol and 2-nitrophenol a yellow colour and thymolphthalein a blue colour. 4-Methylumbelliferone and fluorescein are intensely fluorescent products. xcex1-Naphthol will react with diazonium salts to give highly coloured dyes when released from the substrate. 2-Hydroxy-5-nitropyridine, 6-hydroxynicotinamide and 2-hydroxypyridine-4-carboxamide are further compounds which when released from a substrate are readily measurable when the xe2x80x94OH is ionised. Intensity of colour or fluorescence increases on making the hydrolysate alkaline to fully ionise the phenolic groups for some of the above compounds as is well known in the art.
A number of the preferred substrates yield a Yxe2x80x94OH which is a nitrophenol, or a hydroxypyridine. Preferred substrates include the xcex2-D-ribofuranosides of 2-nitrophenol, 4-methyl umbelliferone, 3- and 4-hydroxypyridine, 2-hydroxy-5-nitropyridine, luminol, 6-hydroxynicotinamide and 4-formylphenol.
A particularly preferred substrate is p-nitrophenyl xcex2-D-ribofuranoside (xe2x80x9cnitrophenylribosidexe2x80x9d). Another particularly preferred substrate is 4-pyridyl xcex2-D-ribofuranoside (xe2x80x9c4-pyridylribosidexe2x80x9d).
The enzyme assay/detection method may be used in a manner which takes advantage of the fact that the chromogenic substrates are considerably more efficiently hydrolysed by some nucleoside hydrolases than others.
In another aspect of the invention chromogenic substrates are hydrolysed by parasite nucleoside hydrolases releasing the chromophore allowing the detection or assay of the parasite especially in mammalian samples. Among the parasites which may be detected/assayed by this type of method are Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia, Herpetomonas, and Leptomonas, especially Trypanosoma. Also especially preferred for use in this method are Giardia. Toxoplasma and Neophora may also be detected/assayed by the method of the invention. As will be apparent to those skilled in the art, the method can be advantageously applied with any parasite containing one or more nucleoside hydrolases which can catalyse hydrolysis of the chromogenic substrate efficiently enough to be detectable/measurable in a mammalian sample.
Trypanosoma cruzi, Giardia intestinalis, and Trypanosoma vaginalis are particularly suitable for assay/detection by the method of the invention.
In another aspect of the invention, deoxyribonucleoside hydrolases and protozoan parasites containing deoxyribonucleoside hydrolases can be detected by analogous methods when the chromogenic substrates used contain a 2-deoxy-xcex2-D-ribofuranosyl moiety rather than a xcex2-D-ribofuranosyl moiety. In preferred embodiments of this aspect the chromogenic substrate is a compound of formula I wherein Xxe2x95x90H. The chromogenic group Y is as defined under formula I. Preferably the chromophore when released from the substrate is selected from one of phenolphthalein, p-nitrophenol, 4-methylumbelliferone, xcex1-naphthol, thymolphthalein. 2-nitrophenol, 2-hydroxy-5-nitropyridine, 6-hydroxynicotinamide, 2-hydroxypyridine-4-carboxamide and fluorescein, especially p-nitrophenol and 4-methylumbelliferone.
Chromogenic substrates may be prepared starting with a ribosyl ester such as 1-O-acetyl-2,3,5-tri-O-benzoyl-xcex2-D-ribofuranose which is commercially available from Aldrich. This can be coupled to a chromophore molecule with a free OH. This may be achieved using a Lewis Acid such as BF3.(OEt)2 as catalyst to give a 1-O-chromogen derivative. The 1 -O-chromogen-triesterified-ribose compound (eg 1-O-chromogen-tri-O-benzoyl ribose) can be extracted and purified and the ester (eg benzoate) groups removed by hydrolysis (eg by stirring overnight in methanol adjusted to about pH 10 with aqueous sodium hydroxide). If so desired, phosphorylation at the five position of the ribofuranose moiety may be accomplished by reaction with N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine in tetrazole-acetonitrile followed by oxidation with m-chloroperoxybenzoic acid. The resulting 5-O-(o-xylylenephosphato)-xcex2-D-ribofuranoside compound may be converted to the corresponding ribofuranoside 5-O-phosphate by hydrogenation e.g. over Pd/C in ethanol followed by neutralisation with aqueous sodium hydroxide.
Reaction scheme 1 includes the above general reaction scheme as Method A.
Alternative syntheses are also possible, for example, by Michael or Koenigs-Knorr type synthesis involving O-protected ribofuranosyl halides (eg acetates, benzoates) and reaction with phenolate salt or phenol and heavy metal catalyst eg Ag(I), Hg(II) salts or oxide. The halide may be a chloride, bromide or a fluoride (eg see Method B of Reaction Scheme 1). 
Reagents:
(Method A): i, Yxe2x80x94OH, BF3.OEt,; ii, N aOH, H2O, MeOH; (Method B); iii, TiCi4, CH2Cl2; iv, Yxe2x80x94Oxe2x88x92Ag+, Toluene; v, K2CO3, MeOH then Amberlite IRC-50 (H+) resin; vi, N,N-diethyl-1,5-dianhydro-2,4,3-benzodioxaphosphepin-3-amine, 1H-tetrazole, then MCPBA; vii, H,, Pd/C, EtOH, then NaOH.
Yxe2x80x94OH or its silver salt (Yxe2x80x94Oxe2x88x92Ag+) is a chromophore as defined in the specification. 
Reagents:
i, MeOH, HCl; ii, 4-MeBzCl, Pyridine; iii, HCl, AcOH; iv, Yxe2x80x94O""Na+, DMF; v, K2CO3, MeOH; vi, N,N-diethyl-1,5-dianhydro-2,4,3-benzodioxaphosphepin-3-amnine, 1H-tetrazole, then MCPBA; vii, H2, Pd /C, EtOH, then NaOH.
Yxe2x80x94Oxe2x88x92Na+ is the sodium salt of Yxe2x80x94OH, a chromophore as defined in the specification.
Corresponding deoxy-xcex2-ribofuranosyl compounds may be prepared using the above methods but with the ribofuranosyl starting materials replaced with the corresponding 2-deoxyribofuranosyl compounds. A preferred method is shown in reaction scheme 2.
Generally in the method of the invention for detection/assay of parasites, the samples to be tested are brought into contact with the substrate at an appropriate pH and the reaction is allowed to proceed. The reaction may take place in a spectrophotometer cuvette to which the chromogenic substrate has been added in an appropriate buffer. The choice of buffer and pH will be influenced by a number of factors including the pH activity profile of the enzyme and the particular parasites under investigation. The choice of buffer and pH can readily be determined by those skilled in the art. The pH will generally be in the range 7-8.5. When a substrate such as nitrophenylriboside or phenolphthalein riboside is being used, use of slightly alkaline pH values may be preferred as, when the chromophore is released, p-nitrophenol and phenolphthalein will then be more coloured. Where a chromophore is coloured at the pH of the reaction its release from the riboside can be directly monitored in a spectrophotometer for example. Where the reaction is carried out at a pH in which the chromophore is not coloured, it may be necessary to allow the reaction to proceed for some time and then adjust the pH to observe the chromophore at a pH where it is coloured.
In a preferred method the sample is brought into contact with p-nitrophenylriboside at about pH 8 and the reaction is monitored by appearance of colour of a nitrophenolate ion formation. The colour may be monitored visually or by measuring absorption at 400 nm.
Where the chromophore is xcex1-napthol, the reaction is allowed to proceed and then after a time the chromophore is reacted with a diazonium salt to produce the coloured compound which is subsequently used to measure the extent of the reaction.
Where the chromophore is luminol, the luminol released in the enzyme reaction is subsequently oxidized by chemical on enzymic means known in the art and the emitted light from the chemiluminescence obtained is measured.
The invention may be practised with many variations so long as the critical feature of a substrate of a nucleoside hydrolase or phosphorylase, where the base is replaced by a chromogenic group, is present. Generally the chromogenic group is not coloured when present on the substrate but is when released by the action of the enzyme or else is otherwise readily detected once liberated from the substrate. Those skilled in the art will appreciate that this may be carried out with many variations.
The reaction need not necessarily be carried out in a test tube or cuvette. The reagents may be included in test strips whereby biological sample is added to the test strip and colour is generated if the sample contains parasites containing one or more enzymes which hydrolyse the chromogenic substrate. For convenience the test strip may form part of a dipstick. Such dipsticks may be prepared according to methods well known in the art.
The invention may be carried out with many sample types, e.g. biological samples including blood or serum samples. The samples may be taken from a mammal (e.g a human, bovine, pig, goat, sheep or horse) but may also be taken from other species (eg fish species or from the environment).
For some samples minimal preparation is necessary. For example the assay for Trypanosoma cruzi, the causative agent of Chagas disease, may be carried out on a blood lysate containing one microlitre of infected blood. The lysate may be prepared by mixing blood with an equal volume of 1% nonionic detergent. Other sample preparation methods may be used. For example samples may be disrupted by use of sonication.
In another aspect of the invention, advantage can be taken of the different substrate specificity of the different parasite enzymes to assist in determining the nature of a parasite infection. Differences in specificities are illustrated in the Examples by comparison of kinetic data from IU-nucleoside hydrolase from Crithidia fasciculata and IAG-nucleoside hydrolase from Trypanosoma brucei brucei. The compound p-nitrophenylriboside is a particularly good substrate for the former but not the latter. In contrast, 4-pyridyl riboside and 2-(5-nitropyridyl) riboside show lower activity with the former enzyme than does nitrophenylriboside, but substantially greater activity with the latter than does the nitrophenylriboside.
Kits containing a chromogenic substrate of Formula (I) optionally together with a buffer and/or materials for processing the biological sample prior to assay/detection (eg a cell-lysing solution) are included within the invention. Preferably the substrate is in a dry form to minimise hydrolysis during storage.